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. 2017 May 24;12:15.
doi: 10.1186/s13020-017-0136-y. eCollection 2017.

Chemical Constituents From a Gynostemma laxum and Their Antioxidant and Neuroprotective Activities

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Free PMC article

Chemical Constituents From a Gynostemma laxum and Their Antioxidant and Neuroprotective Activities

Ji Yeon Seo et al. Chin Med. .
Free PMC article

Abstract

Background: A few bioactivities of constituents from Gynostemma laxum, which has been collected in Vietnam, have been reported until now. There is no report about the effects of constituents from G. laxum although the nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated heme oxygenase-1 (HO-1) antioxidant defense system is involved in neuroprotection in the brain. Therefore, we investigated whether quercetin (2), benzoic acid (10) and their analogues (1, 3-9 and 11) from G. laxum have the antioxidant and neuroprotective activities and also their underlying mechanism.

Methods: To examine their neuroprotective and antioxidant activities, oxytosis, total oxidant scavenging capacity (TOSC), 2,7-dichlorofluorescein (DCFDA), dihydroethidium (DHE), antioxidant response element (ARE)-luciferase reporter gene assays, Western blot analysis, real time-PCR, immunocytochemistry and in silico 3D molecular docking simulation were performed.

Results: The study of constituents using chromatographic techniques and spectroscopic analysis showed that G. laxum contained an abundance of quercetin (2), benzoic acid (10) and their analogues (1, 3-9 and 11). Our data demonstrated that quercetin (2) and its analogue (4) among the constituents from G. laxum showed the strongest neuroprotective effect against oxytosis triggered by the excessive amount of glutamate. Compounds 2, 4, 6 and 11 exhibited reactive oxygen species (ROS) inhibitory and ARE transcriptional activities in immortalized hippocampal HT22 cell line. Among them, compound 4, a second active compound, induced Nrf2/HO-1 activation. They were also fit stable onto the Tramtrack and Bric-à-Brac (BTB) domain of Kelch-like ECH-associated protein 1 (Keap1), a known Nrf2 inhibitor protein, based on the results of docking and interaction energies. Overall, these data suggest that -OH and -OCH3 groups of quercetin and its analogues are responsible for their neuroprotective effect.

Conclusions: In summary, the major constituents of G. laxum had strong antioxidant and neuroprotective activities so that they could consider as a natural antioxidant supplement. Furthermore, G. laxum might be used beneficially in reducing oxidative complications with the further deep investigation in vivo.

Keywords: Antioxidant; Gynostemma laxum; Keap1; Neuroprotection; Quercetin analogues.

Figures

Fig. 1
Fig. 1
Chemical structures of isolated compounds
Fig. 2
Fig. 2
Neuroprotective effects of isolated compounds from G. laxum in HT22 cells. a Cells were treated with each isolated compound from G. laxum at 10 or 20 μM in the presence or absence of 10 mM glutamate for 12 h. Values expressed to AV ± SD. N = 3, the significance was presented as ***p < 0.001 compared to the control, ### p < 0.001 compared to the glutamate-treated cell group. b Cells were treated with compounds 2, 4, 6, and 11 at 1, 5, 10, and 20 μM in the presence of 10 mM glutamate for 12 h. Values expressed to AV ± SD. N = 3, the significance was presented as ***p < 0.001 compared to the control, # p < 0.05 and ### p < 0.001 compared to the glutamate-treated cell group
Fig. 3
Fig. 3
Effects of compounds 2, 4, 6, and 11 on ROS inhibitory and ARE transcription inducing activities. a DCFDA and DHE assays were visualized as the bright field cell images, intracellular ROS and superoxide anion levels by compounds 2, 4, 6, and 11 at 10 μM for 10 h plus 10 mM glutamate. The magnification is ×200. b ARE-luciferase reporter gene assay was performed using HT22-ARE cells. Cells were treated with compounds 2, 4, 6, and 11 at 5, 10, and 20 μM for 16 h. The specific methods are described in “Methods” section. Values expressed to AV ± SD. N = 3, the significance was presented as *p < 0.05 and ***p < 0.001 compared to the control
Fig. 4
Fig. 4
Nrf2-mediated HO-1 induction by compound 4 in HT22 cells. a Nuclear Nrf2 accumulation by compound 4 at 5, 10, and 20 μM was analyzed by Western blotting. b Translocation of Nrf2 into nucleus by compound 4 at 20 μM was determined by the immunocytochemistry procedure using confocal fluorescence microscope. c Real-time PCR evaluated the mRNA expression of HO-1 by treatment of compound 4 at 5, 10, and 20 μM for 24 h. Values expressed to AV ± SD. N = 3, the significance was presented as *p < 0.05 compared to the control. d HO-1 expression pattern by treatment of compound 4 at 5, 10, and 20 μM for 24 h. Values expressed to AV ± SD. N = 2, the significance was presented as *p < 0.05 compared to the control
Fig. 5
Fig. 5
Molecular docking and interaction images of compounds 2 and 4 with Keap1. a Quercetin docked in BTB domain of Keap1 (left-up side) and mutant BTB domain at C151W of Keap1 (right-up side). The interpolated structures were shown in this figure. The 2D diagram of ligand interactions was illustrated at down side. b Quercetin-4′-methyl ether docked in BTB domain of Keap1 (left-up side) and mutant BTB domain at C151W of Keap1 (right-up side). The interpolated structures were shown in this figure. The 2D diagram of ligand interactions was illustrated at down side

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